Human papilloma viruses are small (approximately 8 kb) DNA viruses and more than 40 types have now been reported as inducing epithelial or fibroepithelial proliferations of the skin or mucosa (zur Hausen, H., 1977, Current Topics in Microbiol. and Immunol. 78, 1-30). In particular the DNA of several HPV types is found in a variety of genital lesions, ranging from benign warts (both common and genital) which often contain HPV 6b or HPV 11 DNA (Gissmann L. et al, 1983, Proc. Nat. Acad. Sci. (USA) 80, 560-563), to invasive squamous cell carcinomas of the cervix, which frequently harbour HPV -16, -18, -33 or -35 genomes (Durst M., et al 1983, Proc. Nat. Acad. Sci. USA 80, 3812-3815; Beaudenon S., et al 1986, Nature, Vol. 321, 246-249).
From the cytological examination of cervical smears it is possible to designate approximately 10% of the women from whom the smear samples were taken as being infected with HPV. Such women are then referred for further physical examinations, which frequently involve invasive diagnostic procedures. These further examinations usually reveal that of that 10%, most women are suffering from a non-serious HPV infection, whilst in a few, the forms of HPV infection will be more ominous. Thus in a screening system it would be useful to have a diagnostic test which both identifies HPV infection as being present, and distinguishes non-serious from serious HPV infection. In order to minimize equipment and training costs it would be advantageous for any new diagnostic test to utilize existing technology and slot easily into the present screening procedure. Furthermore, the provision of objective answers (rather than subjective assessments) by any such tests would allow automation, and this in turn would help to reduce both costs and waiting times, the latter being frequently stressful for the patients concerned.
To date no antibody-based clinical diagnostic tests are available for the detection of HPV infection. This is largely due to two problems. Firstly, it has not been possible to date to establish a permissive tissue culture system for the in vitro propagation of papilloma viruses. Secondly, clinical material is difficult to obtain, the viral proteins are usually present in only very small quantities in the clinical lesions, and the HPV causing the lesion is generally of unknown type. These two factors mean that a reliable and sufficient source of typed (i.e. known) HPV is not available for use as both an immunogen and screening agent in the large-scale production of monoclonal antibodies.
Up until now the production of antibodies to HPV (for research purposes) has been achieved using HPV proteins expressed in bacterial expression systems, or in the form of synthetic oligopeptides, as immunogens, and screening the hybridoma supernatants for antibodies produced against the immunogens by reusing the immunogens or various combinations thereof (Banks L., et al 1987, J. Gen. Virol. Vol 68, 3081-3089; Doorbar J. and Gallimore P., et al 1987, J. Virology 61, p. 2793-2799).
However, these known protocols for the production of monoclonal antibodies to HPV are generally unsuitable for the production of monoclonal antibodies which are to be used in immunocytochemical diagnostic tests for screening procedures. This is because antibodies produced by these protocols will not necessarily react with the naturally occurring HPV protein in infected human cells. In addition, the epitopes recognized by these antibodies will not necessarily be those epitopes which are resistant to the standard procedures involved in the sampling, fixing and storing of clinical specimens.
For successful use of an antibody in a diagnostic test the antibody must recognize an epitope which is common to the immunogen and the test sample as prepared for analysis (i.e. after any pre-treatment of tissues such as cryopreservation, sectioning and fixing). Therefore, the system chosen for screening large numbers of hybridoma culture supernatants must be such that it aids selection of diagnostically useful antibodies.